In recent years, oligonucleotides have been a subject of interest in the on-going development of pharmaceutical products called nucleic acid drugs, and particularly, from the viewpoints of high selectivity of target gene and low toxicity, the development of nucleic acid drugs utilizing an antisense method is actively underway. The antisense method is a method of selectively altering the expression of a protein that is encoded by a target gene, by introducing into a cell an oligonucleotide (antisense oligonucleotide (ASO)) which is complementary to a partial sequence of the mRNA (sense strand) of a target gene.
As illustrated in FIG. 1 (upper portion), when an oligonucleotide comprising an RNA is introduced into a cell as an ASO, the ASO binds to a transcription product (mRNA) of the target gene, and a partial double strand is formed. It is known that this double strand plays a role as a cover to prevent translation by a ribosome, and thus the expression of the protein encoded by the target gene is inhibited.
On the other hand, when an oligonucleotide comprising a DNA is introduced into a cell as an ASO, a partial DNA-RNA hetero-duplex is formed. Since this structure is recognized by RNase H, and the mRNA of the target gene is thereby decomposed, the expression of the protein encoded by the target gene is inhibited. (FIG. 1, lower portion). Furthermore, it has been also found that in many cases, the gene expression suppressing effect is higher in the case of using a DNA as an ASO (RNase H-dependent route), as compared with the case of using an RNA.
On the occasion of utilizing an oligonucleotide as a nucleic acid drug, various nucleic acid analogs such as Locked Nucleic Acid (LNA) (registered trademark), other bridged nucleic acids, and the like have been developed in consideration of an enhancement of the binding affinity to a target RNA, stability in vivo, and the like.
Antisense oligonucleotides can be applied to induce exon skipping during the processing of pre-mRNA. The concept is illustrated in FIG. 2. The figure shows a double-stranded DNA segment, and transcription of the DNA yields a pre-mRNA consisting of exons (coding regions) and introns (non-coding regions). Generally, before the mRNA is translated into a peptide (protein) sequence, the cell processes the pre-mRNA to remove the intron regions. It is known that antisense oligonucleotides that target and bind to the pre-mRNA can induce the cell to not include (skip over) an exon. As shown in FIG. 2, the pre-mRNA includes exons 1, 2, 3, and 4. Under normal operation, without an ASO present, the introns would be removed and exons 1, 2, 3, and 4 would be spliced together to yield a full length mRNA.
However, in the presence of an ASO that binds to a target site, illustrated here as being in exon 2, the cell will, in addition to removing the introns, exclude exon 2 to yield a truncated splice-switched mRNA of exons 1, 3, and 4.
As is well-known in the art, exon skipping and splice switching is of interest for treating or ameliorating the effects of genetic mutations. Certain genetic diseases are thought to be treatable at the genetic level by such a mechanism, rather than at the protein level. Two examples are Duchenne muscular dystrophy and spinal muscular dystrophy (Non-Patent Documents 1-4).
Whereas siRNA and gapmer antisense oligonucleotides act to suppress gene expression, splice-switching oligonucleotides (SSOs) act to modify pre-mRNA splicing. Such oligonucleotides can “repair” RNA that would otherwise not be processed correctly, or, they can induce the formation of novel proteins. Because splice variant proteins constitute a large portion of the proteins in humans, the ability to induce and/or modulate splice-switching is the subject of great interest.
Oligonucleotides used as antisense agents usually contain modified nucleotides or nucleotide analogues in order to enhance binding affinity to the targeted sequence. As illustrated in FIG. 3, since the sugar moiety of a natural nucleic acid (RNA or DNA) has a five-membered ring with four carbon atoms and one oxygen atom, the sugar moiety has two kinds of conformations, an N-form and an S-form. It is known that these conformations swing from one to the other, and thereby, the helical structure of the nucleic acid also adopts different forms, an A-form and a B-form. Since the mRNA that serves as the target of the aforementioned ASO adopts a helical structure in the A-form, with the sugar moiety being mainly in the N-form, it is important for the sugar moiety of the ASO to adopt the N-form from the viewpoint of increasing the affinity to RNA. A product that has been developed under this concept is a modified nucleic acid such as a LNA (2′-O,4′-C-methylene-bridged nucleic acid (2′,4′-BNA)). For example, in the LNA, as the oxygen at the 2′-position and the carbon at the 4′-position are bridged by a methylene group, the conformation is fixed to the N-form, and there is no more fluctuation between the conformations. Therefore, an oligonucleotide synthesized by incorporating several units of LNA has very high affinity to RNA and very high sequence specificity, and also exhibits excellent heat resistance and nuclease resistance, as compared with oligonucleotides synthesized with conventional natural nucleic acids (see Patent Document 1). Since other artificial nucleic acids also have such characteristics, much attention has been paid to artificial nucleic acids in connection with the utilization of an antisense method and the like (see Patent Documents 1 to 7).
Furthermore, when an oligonucleotide is applied to a drug, it is important that the relevant oligonucleotide can be delivered to the target site with high specificity and high efficiency. Cell-penetrating peptides, such as the short, positively-charged arginine-rich peptides P007 and B peptide, can improve the uptake of oligonucleotides into cells when conjugated to the oligonucleotide (Non-Patent Document 5). Even if an oligonucleotide enters a cell, for it to have a splice-switching effect the oligonucleotide needs to enter the nucleus. Delivery of a splice-switching oligonucleotide across the nuclear membrane remains a challenge (Non-Patent Document 6). It is an object of the invention to provide double-stranded nucleic acid agents that provide enhanced delivery of antisense oligonucleotides into cell nuclei. It is a further object of the invention to provide oligonucleotides that provide enhanced levels of exon skipping and/or alternative spliced processing of pre-mRNA.
In addition, as methods for delivering an oligonucleotide to certain body regions, a method of utilizing lipids such as cholesterol and vitamin E (Non-Patent Documents 7 and 8), a method of utilizing a receptor-specific peptide such as RVG-9R (Non-Patent Document 9), and a method of utilizing an antibody specific to the target site (Non-Patent Document 10) have been developed.